The AAA+ (ATPases associated with diverse cellular activities) superfamily of enzymes couples ATP hydrolysis with the generation of mechanical force to regulate diverse aspects of prokaryote and eukaryote biology. These complex proteins typically form ring-shaped hexamers with a central pore, and ATP-dependent conformational changes that propagate through these molecular machines can promote DNA replication, the disassembly of membrane-fusing complexes during organelle biogenesis and vesicular transport, the trafficking of cellular cargos along microtubules, and the unfolding of proteins for proteolysis. One of the subclasses of AAA+ ATPases includes dynein 1 and dynein 2. Cytoplasmic dynein 1 acts in concert with dynactin and the nuclear protein NuMA to crosslink and focus the minus ends of microtubules within the mitotic spindle. These actions create the canonical fusiform shape and localize γ-tubulin-containing, microtubule-nucleating complexes to the spindle poles. Cytoplasmic dynein 1 inhibition, by blocking antibodies or dominant negative constructs, disrupts mitotic spindle assembly, resulting in splayed microtubule ends and reduced γ-tubulin recruitment. Dynein 2 is integral in protein trafficking mechanisms within the primary cilium, where it is involved in moving macromolecules along the axoneme. Intraflagellar retrograde trafficking, utilizes cytoplasmic dynein 2 and the IFTA complex. Small-molecule AAA+ ATPase inhibitors, particularly those that can act rapidly and reversibly are therefore much needed, both as probes of dynein function and as potential antitumor agents.